Sónia Andreia
Oliveira Santos
Compostos fenólicos a partir de subprodutos da
indústria florestal
Phenolic compounds from forest industrial
by-products
2012
Sónia Andreia
Oliveira Santos
Compostos fenólicos a partir de subprodutos da
indústria florestal
Phenolic compounds from forest industrial
by-products
Tese apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Química, realizada sob a orientação científica do Doutor Carlos Pascoal Neto, Professor catedrático do Departamento de Química da Universidade de Aveiro e do Doutor Armando Jorge Domingues Silvestre, Professor associado com agregação do Departamento de Química da Universidade de Aveiro
Apoio financeiro do projeto AFORE (CP-IP 228589-2) no âmbito do VII Quadro Comunitário de Apoio.
Apoio financeiro do projeto WaCheUp (NMP-TI-3 – STRP 013896) no âmbito do VI Quadro Comunitário de Apoio.
Apoio financeiro da FCT e do FSE (SFRH / BD / 42021 / 2007), no âmbito do Programa Operacional Potencial Humano (POPH) do QREN.
o júri
presidente Doutor Carlos Alberto Diogo Soares Borrego
professor catedrático da Universidade de Aveiro
Doutor Carlos Pascoal Neto
professor catedrático da Universidade de Aveiro
Doutor Nuno Filipe da Cruz Batista Mateus
professor associado com agregação da Faculdade de Ciências da Universidade do Porto
Doutor Armando Jorge Domingues Silvestre
professor associado com agregação da Universidade de Aveiro
Doutora Maria do Rosário Reis Marques Domingues
professora auxiliar da Universidade de Aveiro
Doutora Paula Cristina de Oliveira Rodrigues Pinto
agradecimentos Os meus agradecimentos vão, em particular, para o Professor Doutor Armando J. D. Silvestre e Professor Doutor Carlos Pascoal Neto, pela excelente
orientação científica, apoio incondicional e, principalmente, por toda a confiança que depositaram em mim.
À Professora Doutora Maria Rosário Domingues, agradeço todo o apoio e entusiasmo que me deu ao longo destes anos e de forma incansável. Agradeço também a preciosa ajuda na espectrometria de massa.
Gostaria de agradecer ao Professor Doutor Carlos Silva por todo o apoio na extração supercrítica e pela confiança que depositou em mim.
À Doutora Paula Pinto faço um especial agradecimento por tudo o que me transmitiu no início deste trabalho, por toda a sua amizade e, acima de tudo, pela sua sempre boa disposição.
À Doutora Carmen Freire agradeço todo o apoio, os conselhos e incentivos. Gostaria de agradecer ao Doutor Juan Jose Villaverde por todo o apoio, amizade e incentivo que me facultou, sempre de forma incansável. Ao grupo de espectrometria de massa do Departamento de Química da Universidade de Aveiro gostaria de fazer um especial agradecimento por toda a sua disponibilidade, em particular, ao Professor Doutor Pedro Domingues, pela preciosa ajuda com o HPLC.
Gostaria de agradecer a todos os meus colegas de trabalho, aos dos “nossos” laboratórios e aos dos laboratórios ao lado, aos que ainda lá trabalham e aos que já foram. Todos tornaram todos estes anos tempos excecionais.
Ao Ricardinho, à Carla, à Andreinha, ao Rui, à Lili, à Mónica, à Bi, à Gi e à Belinda, faço um especial agradecimento, por terem tornado todo este percurso muito mais fácil.
A todos os que me ouvirem dizer infinitas vezes “Good morning, my name is Sónia…”, agradeço a enorme paciência. À Nor, ao Tiago, ao Fernando, à Vera, ao Ricardo, ao Nelson, à Vânia e à Joana, que de forma incansável sempre me apoiaram e animaram, faço um especial agradecimento. Agradeço, acima de tudo, por compreenderem tantas ausências. Ao João Paulo, agradeço o facto de me ter transmitido ao longo destes anos que com força de vontade nenhum obstáculo é suficientemente grande.
Por último, mas não menos importante, agradeço à minha família, em particular aos meus pais e irmãos por todo o apoio e principalmente por compreenderem quando eu não pude estar. Agradeço especialmente ao Tiago, por inúmeras razões, entre as quais todo o incansável apoio,
palavras-chave Compostos fenólicos, Quercus suber L., cortiça, Eucalyptus globulus Labill., Eucalyptus grandis, Eucalyptus urograndis (Eucalyptus grandis x Eucalyptus urophylla), Eucalyptus maidenii, eucalipto, HPLC-MS, atividade antioxidante, extração supercrítica.
resumo Em Portugal, as indústrias corticeira e de pasta de papel constituem um importante sector económico, contudo, gerando elevadas quantidades de subprodutos. Estes subprodutos poderiam ser explorados em aplicações de alto valor acrescentado, como fonte de compostos fenólicos, por exemplo, em vez de serem apenas queimados para produção de energia. Estes compostos são conhecidos pelas suas inúmeras propriedades, entre as quais,
antioxidante, anti-inflamatória e anti-trombótica.
Neste estudo as frações fenólicas da maior parte dos subprodutos gerados nas indústrias corticeira e de pasta de papel foram caracterizados em detalhe, com vista à sua valorização. A fração fenólica das cascas de Eucalyptus globulus, E. grandis, E. urograndis e E. maidenii, bem como da cortiça de Quercus suber e resíduos provenientes da sua exploração, nomeadamente, o pó de cortiça e os condensados negros, foi obtida por processos convencionais de extração sólido-líquido.
No caso da casca de E. globulus, foi ainda avaliado o potencial de metodologias “verdes” no processo de extração de compostos fenólicos, usando extração com CO2 supercrítico. Esta técnica foi otimizada com recurso
a metodologias de superfície de resposta.
Na identificação e quantificação dos compostos fenólicos foi usada
cromatografia líquida de alta resolução aliada a técnicas de espectrometria de massa. O teor de fenólicos totais foi ainda determinado pelo método de Folin-Ciocalteu, essencialmente para efeitos comparativos. A caracterização da fração fenólica de cada extrato foi ainda complementada com a análise da atividade antioxidante, usando o radical 2,2-difenil-1-picrilhidrazilo (DPPH). Foram identificados trinta compostos fenólicos na casca de E. globulus, 17 deles referenciados pela primeira vez como seus constituintes, nomeadamente os ácidos quínico, di-hidroxifenilacétic, cafeico e metil-elágico,
bis-hexa-hidroxidifenoil(HHDP)-glucose, galoil- bis-HHDP-glucose, galoil-HHDP-glucose, isoramnetina—hexosídeo, quercetina-hexosídeo, ácido metil-elágico-pentosídeo, miricetina-ramnosídeo, isoramnetina-ramnosídeo, mearnsetina, floridzina, mearnsetina-hexosídeo, luteolina e uma proantocianidina B. Neste trabalho, foi estudada pela primeira vez a composição fenólica das cascas de E. grandis, E. urograndis e E. maidenii. (cont.)
resumo (cont.)
Treze, doze e vinte e quatro compostos fenólicos foram identificados nas cascas de E. grandis, E. urograndis e E. maidenii, respetivamente. Entre estes compostos encontram-se os ácidos quínico, gálico, metilgálico,
protocatequínico, clorogénico e elágico, catequina, galoil-bis-HHDP-glucose, digaloilglucose, epicatequina, quercetina-glucoronídeo,
di-hidroxi-isopropilcromona-hexosídeo, isoramnetina-hexosídeo, ácido elágico-ramnosídeo, taxifolina, quercetina-hexosídeo,
di-hidroxi-(metilpropil)isopropilcromona-hexosídeo, ácido metil-elágico-pentosídeo, miricetina-ramnosídeo, isoramnetina-ramnosídeo, aromadendrina-ramnosídeo, mearnsetina, mearnsetina-hexosídeo, eriodictiol, quercetina, isoramnetina e naringenina. A análise da fração fenólica da cortiça permitiu identificar vinte e dois compostos fenólicos, dez deles referenciados pela primeira vez como seus constituintes, nomeadamente, os ácidos quínico, salicílico, p-hidroxifenil-lático e metilgálico, ácido carboxílico da brevifolina, eriodictiol, naringenina, um éster isoprenílico do ácido cafeico, isoramnetina-ramnosídeo e isoramnetina. No pó de cortiça industrial foram identificados dezasseis compostos fenólicos, nomeadamente os ácidos quínico, gálico, protocatequínico, cafeico, ferúlico, elágico e metilgálico, esculetina, ácido carboxílico da brevifolina,
coniferaldeído, um éster isoprenílico do ácido cafeico, uma dilactona do ácido valoneico, ácido elágico-pentosídeo, ácido elágico-ramnosídeo, isoramnetina-ramnosídeo e isoramnetina. Destes, apenas o ácido elágico foi previamente referenciado como componente do pó de cortiça. Do mesmo modo, treze compostos fenólicos foram identificados no condensado negro, doze deles referenciados pela primeira vez como seus constituintes. São eles os ácidos quínico, gálico, p-hidroxifenil-láctico, protocatequínico, p-coumarico, cafeico e elágico, vanilina, esculetina, coniferaldeído, um éster isoprenílico do ácido cafeico e o eriodictiol.
A extração supercrítica de compostos fenólicos da casca de eucalipto permitiu não só verificar os parâmetros que afetam a qualidade e quantidade finais dos extratos, como também obter os valores ótimos para estes parâmetros. Esta extração mostrou ainda ser bastante seletiva para determinados grupos de compostos fenólicos, como as flavanonas eriodictiol e naringenina e para o flavonol O-metilado isoramnetina.
Este é também o primeiro estudo envolvendo a determinação da atividade antioxidante de extratos da cortiça e dos resíduos da sua exploração, bem como da casca de E. grandis, E. urograndis e E. maidenii.
A vasta gama de compostos fenólicos identificados em cada extrato analisado, assim como as prominentes atividades antioxidantes, todas na mesma gama de valores do bem conhecido antioxidante comercial, ácido ascórbico, são claramente um grande contributo para a valorização destes subprodutos
keywords Phenolic compounds, Quercus suber L., cork, Eucalyptus globulus Labill., Eucalyptus grandis, Eucalyptus urograndis (Eucalyptus grandis x Eucalyptus urophylla), Eucalyptus maidenii, HPLC-MS, antioxidant activity, supercritical extraction.
abstract In Portugal, the cork and the pulp and paper industries are important economic sectors, however, generating substantial amounts of products. These by-products could be exploited in added value applications, rather than being simply burned for energy production, as, for example, as a source of the valuable phenolic compounds. These compounds are known by their innumerous properties, as antioxidant, inflammatory or even anti-thrombotic.
In this study, the phenolic fractions of the most abundant cork and pulp
industrial residues were characterised in detail, aiming at up-grading them. The phenolic fraction of the barks of Eucalyptus globulus, E. grandis, E. urograndis and E. maidenii as well as the cork from Quercus suber and the residues of its exploitation, namely, cork powder and black condensates, were obtained by conventional solid-liquid extractions.
In the case of E. globulus bark, the potential application of green
methodologies in the extraction of phenolic compounds was also evaluated, by using supercritical CO2 extraction. This approach was optimized by using
surface response methodology.
High-performance liquid chromatography coupled with mass spectrometry techniques were used in the identification and quantification of phenolic compounds. The total phenolic content was also accessed by the
Folin-Ciocalteu method, mainly for comparative purposes. The characterization of the phenolic fraction of each extract was also complemented with antioxidant activity measurements, by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging.
Thirty phenolic compounds were identified as constituents of E. globulus bark, 17 of them referenced for the first time, namely, quinic, dihydroxyphenylacetic, and caffeic acids, bis-hexahydroxydiphenoyl(HHDP)-glucose, galloyl-bis-HHDP-glucose, galloyl-galloyl-bis-HHDP-glucose, isorhamentin-hexoside, quercetin-hexoside, methyl-ellagic acid, methyl-ellagic acid (EA)-pentoside, myricetin-rhamnoside, isorhamnetin-myricetin-rhamnoside, mearnsetin, phloridzin, mearnsetin-hexoside, luteolin and a proanthocyanidin B-type dimer.
The phenolic composition of E. grandis, E. urograndis and E. maidenii bark was studied in this work for the first time. Thirteen, twelve and twenty four phenolic compounds were identified in E. grandis, E. urograndis and E. maidenii bark extracts, respectively. These compounds include quinic gallic, protocatechuic, chlorogenic and ellagic acids, methyl gallate, catechin, galloyl-bis-HHDP-glucose, digalloylgalloyl-bis-HHDP-glucose, epicatechin, quercetin-glucuronide, dihydroxy-isopropylchromone-hexoside, isorhamnetin-hexoside, ellagic acid-rhamnoside, taxifolin, quercetin-hexoside,
dihydroxy-(methylpropyl)isopropylchromone-abstract (cont.)
The analysis of the phenolic fraction of cork allowed to identify twenty two phenolic compounds, ten of them reported for the first times as its constituents, namely, quinic, salicylic and p-hydroxyphenyl-lactic acids, eriodictyol,
naringenin, methyl gallate, brevifolin carboxylic acid, caffeic acid isoprenyl ester, isorhamnetin-rhamnoside and isorhamnetin. It were identified sixteen phenolic compounds in industrial cork powder, namely, quinic, gallic,
protocatechuic, caffeic, ferulic and ellagic acids and methyl gallate, esculetin, brevifolin carboxylic acid, coniferaldehyde, caffeic acid isoprenyl ester, valoneic acid dilactone, ellagic acid-pentoside, ellagic acid-rhamnoside, isorhamnetin-rhamnoside and isorhamnetin. From these, only ellagic acid was previously reported as constituent of cork powder. Likewise, thirteen phenolic compounds were identified on black condensate, twelve of them for the first time, namely quinic, gallic, p-hydroxyphenyl-lactic, protocatechuic, p-coumaric, caffeic and ellagic acids and vanillin, esculetin, coniferaldehyde, caffeic acid isoprenyl ester and eriodictyol.
The supercritical extraction of phenolic compounds from E. globulus bark allowed to verify the parameters affecting the qualitatively and quantitatively the final extracts. The optimal conditions of those parameters were obtained. This technique showed to be selective to restrict classes of compounds, such as flavanones and O-methylated flavonols.
This was also the first study involving the evaluation of the antioxidant activity of the phenolic extracts of E. grandis, E. urograndis and E. maidenii bark as well as of cork and the residues of their exploitation.
The vast range of phenolic compounds identified in each vegetal source studied, as well as its outstanding antioxidant activities, all in the same range of the well known commercial antioxidant ascorbic acid, are, clearly, a contribute to the up-grading of these industrial by-products.
Table of contents
ABBREVIATIONS/ACRONYMS ... VII
1 INTRODUCTION ... 1
1.1 BIOREFINERY CONTEXT ... 3
1.1.1 Biorefinery concept ... 3
1.1.2 Biorefinery classification ... 3
1.1.3 Perspectives to the future ... 4
1.2 PORTUGUESE AGRO-FOREST INDUSTRIES TO EXPLOIT WITHIN THE BIOREFINERY CONTEXT ... 5
1.3 AIMS AND SCOPE ... 6
1.4 OUTLINE OF THIS THESIS ... 7
REFERENCES ... 8
2 BIBLIOGRAPHIC REVIEW ... 13
PART A - QUERCUS SUBER L. CORK AND EUCALYPTUS BARK ... 15
2.1 CORK AND PULP AND PAPER INDUSTRIES ... 17
2.1.1 Eucalyptus and pulp and paper processing ... 17
2.1.2 Cork, cork oak and cork processing ... 19
2.2 CORK AND EUCALYPTUS BARK COMPOSITION ... 21
2.2.1 General barks structure ... 21
2.2.2 Eucalyptus bark composition ... 23
2.2.2.1 Polysaccharides ... 24 2.2.2.2 Lignin ... 26 2.2.2.3 Suberin ... 26 2.2.2.4 Inorganic components ... 27 2.2.2.5 Extractives ... 27 2.2.3 Cork composition ... 31 2.2.3.1 Polysaccharides ... 33 2.2.3.2 Lignin ... 34 2.2.3.3 Suberin ... 34 2.2.3.4 Inorganic components ... 36 2.2.3.5 Extractives ... 36
PART B - PHENOLIC COMPOUNDS, EXTRACTION AND ANALYSIS ... 41
2.3 PHENOLIC COMPOUNDS ... 43
2.3.1 Phenolic compounds structures and classification ... 43
2.3.1.1 Phenolic acids and aldehydes ... 44
2.3.1.2 Cinnamic acids ... 45
2.3.1.3 Coumarins, isocoumarins and chromones ... 45
2.3.1.4 Benzophenones, xanthones and stilbenes ... 46
2.3.1.5 Flavonoids ... 46
2.4 PHENOLIC COMPOUNDS EXTRACTION ... 51
2.4.1 Conventional solid-liquid extraction ... 53
2.4.2 Soxhlet extraction ... 54
2.4.3 Accelerated solvent extraction ... 55
2.4.4 Ultrasound-assisted extraction ... 56
2.4.5 Microwave-assisted extraction ... 56
2.4.6 Supercritical fluid extraction ... 57
2.4.7 Solid phase extraction ... 58
2.5 PHENOLIC COMPOUNDS ANALYSIS ... 58
2.5.1 Spectrophotometric analysis ... 58
2.5.1.1 Total phenolic content ... 58
2.5.1.2 Hydrolysable tannins content ... 60
2.5.1.3 Condensed tannins content ... 60
2.5.2 Chromatographic techniques ... 61
2.5.2.1 Size exclusion chromatography... 61
2.5.2.2 Gas chromatography ... 62
2.5.2.3 Thin layer chromatography ... 62
2.5.2.4 High-performance liquid chromatography ... 63
Columns ... 63
Mobile phases ... 64
Detectors ... 65
2.5.2.5 Supercritical fluid chromatography ... 69
2.6 ANTIOXIDANT ACTIVITY ANALYSIS ... 69
2.6.1 Hydrogen atom transfer-based assays ... 71
2.6.1.1 Oxygen radical absorbance capacity ... 71
2.6.1.2 Total peroxyl radical-trapping antioxidant parameter ... 72
2.6.1.3 β-carotene bleaching assay ... 73
2.6.2 Single electron transfer-based assays ... 73
2.6.2.1 Ferric reducing antioxidant power assay ... 73
2.6.2.2 Copper reducing assay ... 74
2.6.2.3 Trolox equivalents antioxidant capacity assay... 74
2.6.2.4 2,2-diphenyl-1-picrylhydrazyl assay ... 75
REFERENCES ... 77
3 EUCALYPTUS GLOBULUS LABILL. BARK AS A SOURCE OF PHENOLIC COMPOUNDS ... 101
PART A - CHARACTERISATION OF PHENOLIC FRACTION OF EUCALYPTUS GLOBULUS LABILL. BARK ... 103
ABSTRACT ... 105
3.1 INTRODUCTION ... 107
3.2.2 Raw material ... 108
3.2.3 Phenolic compounds extraction ... 109
3.2.4 Total phenolic content ... 109
3.2.5 HPLC–UV procedure ... 109
3.2.6 ESI–QqQ–MS analysis ... 110
3.2.7 ESI–IT–MS–MS analysis ... 110
3.2.8 HPLC–UV quantification ... 111
3.3 RESULTS AND DISCUSSION ... 111
3.3.1 Extraction yields and total phenolic content ... 111
3.3.2 Identification of phenolic compounds ... 112
3.3.2.1 Phenolic acids and esters ... 115
3.3.2.2 Flavonoids ... 116
3.3.2.3 Flavonoid glycosides ... 118
3.3.2.4 Ellagic acid and derivatives ... 120
3.3.2.5 Galloylglucose derivatives and ellagitannins ... 121
3.3.3 HPLC quantification of phenolic compounds... 123
3.4 CONCLUSIONS ... 127
REFERENCES ... 127
PART B - SUPERCRITICAL FLUID EXTRACTION OF PHENOLIC COMPOUNDS FROM EUCALYPTUS GLOBULUS LABILL. BARK ... 133
ABSTRACT ... 135
3.5 INTRODUCTION ... 137
3.6 MATERIALS AND METHODS ... 138
3.6.1 Chemicals ... 138
3.6.2 Raw material ... 138
3.6.3 Solid-liquid extractions... 138
3.6.4 Supercritical fluid extraction ... 139
3.6.4.1 Supercritical fluid apparatus ... 139
3.6.4.2 SFE with pure and modified carbon dioxide ... 139
3.6.4.3 SFE design of experiments ... 140
3.6.5 Analytical methods ... 140
3.6.5.1 Extraction yield (EY). ... 140
3.6.5.2 Total phenolic content (TPC) ... 140
3.6.5.3 HPLC-UV procedure ... 140
3.6.5.4 ESI–MSn analysis ... 141
3.6.5.5 HPLC-UV quantification ... 141
3.6.5.6 Antioxidant activity ... 141
3.7 RESULTS AND DISCUSSION ... 142
3.7.1 Preliminary solid-liquid (SLE) and supercritical fluid (SFE) extractions ... 142
3.7.1.3 Phenolic compounds profiles of the supercritical extracts ... 144
3.7.2 Analysis of the designed SFE experiments ... 146
3.7.2.1 Influence of process variables on SFE yield ... 147
3.7.2.2 Influence of process variables on total phenolic content (TPC) ... 148
3.7.2.3 Influence of process variables on total amounts of phenolic compounds quantified by HPLC ... 149
3.7.2.4 Influence of process variables on antioxidant activity ... 150
3.7.2.5 Optimisation of supercritical fluid extraction conditions ... 151
3.8 CONCLUSIONS ... 152
REFERENCES ... 152
4 CHARACTERISATION OF PHENOLIC FRACTION OF EUCALYPTUS GRANDIS, E. UROGRANDIS (E. GRANDIS ×E. UROPHYLLA) AND E. MAIDENII BARKS ... 157
ABSTRACT ... 159
4.1 INTRODUCTION ... 161
4.2 MATERIALS AND METHODS ... 162
4.2.1 Chemicals ... 162
4.2.2 Raw materials ... 162
4.2.3 Phenolic compounds extraction ... 162
4.2.4 Total phenolic content ... 163
4.2.5 HPLC-UV procedure ... 163
4.2.6 ESI–QqQ–MS analysis ... 164
4.2.7 ESI–IT–MS/MS analysis ... 164
4.2.8 HPLC-UV quantification... 164
4.2.9 Antioxidant activity ... 165
4.3 RESULTS AND DISCUSSION ... 165
4.3.1 Extraction yield and total phenolic content ... 165
4.3.2 Identification of phenolic compounds ... 166
4.3.3 HPLC quantification of phenolic compounds... 171
4.3.4 Antioxidant activity ... 175
4.4 CONCLUSIONS ... 175
REFERENCES ... 176
5 CHARACTERISATION OF PHENOLIC FRACTION FROM QUERCUS SUBER L. CORK AND CORK BY-PRODUCTS ... 181
PART A - CHARACTERISATION OF PHENOLIC FRACTION OF QUERCUS SUBER L. CORK ... 183
ABSTRACT ... 185
5.1 INTRODUCTION ... 187
5.2.2 Raw material ... 189
5.2.3 Phenolic compounds extraction ... 189
5.2.4 HPLC–MS analysis ... 189
5.2.5 HPLC-UV quantification... 190
5.2.6 Total phenolic content ... 190
5.2.7 Antioxidant activity ... 190
5.3 RESULTS AND DISCUSSION ... 191
5.3.1 Extraction yields and total phenolic content ... 191
5.3.2 Identification of phenolic compounds ... 192
5.3.2.1 Phenolic acids, aldehydes and derivatives ... 194
5.3.2.2 Cinnamic acids ... 195
5.3.2.3 Coumarins ... 196
5.3.2.4 Flavonoids ... 196
5.3.3 HPLC quantification of phenolic compounds... 197
5.3.4 Antioxidant activity ... 198
5.4 CONCLUSIONS ... 199
REFERENCES ... 199
PART B - PHENOLIC COMPOUNDS FROM INDUSTRIAL CORK BY-PRODUCTS ... 205
ABSTRACT ... 207
5.5 INTRODUCTION ... 209
5.6 MATERIALS AND METHODS ... 210
5.6.1 Chemicals ... 210
5.6.2 Raw materials ... 211
5.6.3 Sample preparation ... 211
5.6.4 Total phenolic content ... 211
5.6.5 HPLC-UV procedure ... 212
5.6.6 ESI–MSn analysis ... 212
5.6.7 HPLC-UV quantification... 212
5.6.8 Antioxidant activity ... 213
5.7 RESULTS AND DISCUSSION ... 213
5.7.1 Extraction yields and total phenolic content ... 213
5.7.2 Identification of phenolic compounds ... 214
5.7.2.1 Phenolic acids, aldehydes and derivatives ... 216
5.7.2.2 Cinnamic acids and derivatives ... 216
5.7.2.3 Coumarins and derivatives ... 218
5.7.3 HPLC quantification of phenolic compounds... 220
5.7.4 Antioxidant activity ... 223
5.8 CONCLUSIONS ... 224
6.1 GENERAL DISCUSSION ... 231 6.2 CONCLUDING REMARKS AND FUTURE WORK ... 241 REFERENCES ... 242
Abbreviations/Acronyms
λmax – wavelength for which a compound has a maximum ultraviolet absorbance AA – antioxidant activity
AAE – ascorbic acid equivalents
AAPH – 2,2‘-azobis(2-amidinopropane) dihydrochloride ABTS – 2,2‘-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) AOP –antioxidant potential
APCI – atmospheric pressure chemical ionisation ASE – accelerated solvent extraction
BC – black condensate
BHA – butylated hydroxyanisole BHT – butylated hydroxytoluene BHQ – tert-butylhydroquinone CE – capillary electrophoresis
CUPRAC – cupric reducing antioxidant capacity CZE – capillary zone electrophoresis
DAD – diode array detector
DPPH – 2,2-diphenyl-1-picrylhydrazyl DNA – deoxyribonucleic acid
EA – ellagic acid ET – electron transfer EtOAc – ethyl acetate EtOH – ethanol
ESI – electrospray ionisation EY – extraction yield
FAB – fast atom bombardment FID – flame ionization detector FL – fluorescence
GAE – gallic acid equivalents GC – gas-chromatography HAT – hydrogen atom transfer HHDP – hexahydroxydiphenoyl
HPCE – high-performance capillary electrophoresis HPLC – high-performance liquid chromatography ICP – industrial cork powder
LCF – lignocellulosic feedstock LOD – limit of detection
LOQ – limit of quantification m/z – mass-to-charge ratio
MAE – microwave-assisted extraction MALDI – matrix assisted laser desortion MeOH – methanol
MS – mass spectrometry
MS/MS – tandem mass spectrometry MSn – multistage mass spectrometry NC – natural cork
NMR – nuclear magnetic ressonance NP – normal phase
ORAC – oxygen radical absorbance capacity PC-HPLC – phenolic content, quantified by HPLC PD – plasma desorption
PLE – pressurized liquid extraction QqQ – triple quadrupole mass analyser RP – reversed-phase
R-PE – R-phycoerythrin
SFE – supercritical fluid extraction SLE – solid-liquid extraction SPE – solid phase extraction SPME – solid phase microextraction TBA – thiobarbituric acid
TEAC – trolox equivalents antioxidant capacity TLC – thin layer chromatography
TPC – total phenolic content, quantified by Folin-Ciocalteu method TPTZ – 2,4,6-tripyridyl-s-triazine
TRAP – total peroxyl radical-trapping antioxidant parameter TSP – thermospray ionisation
ToF – time of flight
TPTZ – 2,4,6-tri(2-pyridyl)-s-triazine
UHPLC – ultra high-performance liquid chromatography US – ultrasounds
USAE – ultrasound-assisted extraction UV – ultraviolet
Chapter 1
1.1
Biorefinery context
Over the last years there is a large increase on the search of new solutions to the inevitable depletion of fossil resources, coupled with the growing interest on environmental concerns. These have been lead to an emerging research trend in the search of bio-based products, within the biorefinery concept [1-6].
1.1.1
Biorefinery concept
“A biorefinery is a facility that integrates biomass conversion processes and equipment to produce fuels, power, and chemicals from biomass”- definition of biorefinery by the National Renewable Energy Laboratory [7]. There are many definitions of this term, however, all of them converging to the same goal: transform different biomass feedstock into useful products by using diverse technologies and processes [1, 4, 8]. Despite, in the last decade, this term has been widely used, this concept is not recent. The novelty is to use biomass (products from agricultural commodities) to produce a wide range of end-products (fuels, power, materials and chemicals), by using complex process as occurs in a petroleum refinery [8]. Ideally, a biorefinery should combine together chemical/biochemical and thermal conversion processes to convert biomass into a wide variety of products as well power for its own use and ideally for exportation to the network [8] Several biomass sources have been focused, such as wood and forest materials (lignocellulosics), agriculture crops and wastes, agro-food industries wastes or aquatic plants [1, 5].
1.1.2
Biorefinery classification
Biorefineries have been classified into phase I, II and III biorefineries, according to the feedstock’s used [4, 8]. A phase I biorefinery has a fixed process capability, and uses only one feedstock as raw material. A dry-milling ethanol plant is an example of this type of biorefineries, which are already considered economically viable. A biorefinery plant, using also grain as feedstock, but with the capability to produce a range of end-products, as the wet-milling technology, is classified as a phase II biorefinery. A phase III biorefinery, the most advanced and flexible, uses different types of feedstock’s and technologies and has the capability to produce various products, as chemicals and/or fuels [4, 8]. Currently three phase III biorefinery systems, classified according to their processed raw materials, have been object of research and development [1, 4]:
wood, straw or corn stover, which are firstly fractionated into their main fractions (hemicelluloses, cellulose, lignin and extractives) and further converted into chemicals or fuels/energy;
Whole-crop biorefinery – uses cereals, as rye or maize as raw material. This type of biorefinery uses the entire plant to produce useful products, materials and energy. The first step involves the separation of the seed from the straw. The seeds may be processed to yield starch or other wide variety of products, while straw is used as raw material in a LCF biorefinery; and
Green biorefinery – uses natural-wet feedstock’s, as green plants and crops in a multi-product system, which handle its fractions and multi-products, according to the physiology of the plant material.
The LCF biorefinery has shown to be the most promising as it has the potential to accommodate a wide range of raw materials at a competitive price (straw, reed, grass, wood, paper-waste, etc.) that can yield conversion products in both the traditional petrochemical-dominated and the future bio-based product markets [2]. Forests are clearly among the most important lignocellulosic sources, and the biorefineries can be developed based on current agro-forest industry facilities. One of the most discussed examples is the implementation of the biorefinery concept based on the existing pulp mills, producing added chemicals and other end-products from biomass residues and pulping waste streams, together with pulp and paper [2, 9-12].
1.1.3 Perspectives to the future
There are several requirements to the successful development of biorefineries in the future. Specific attention should be given to the development and application of industrial biorefinery technologies to become technically and economically viable [3, 9]. Furthermore the biorefining technologies will have to compete with oil based technologies, which have been optimised along the last century [4]. One important aspect is related with the logistic of the flow of feedstock’s, being necessary to achieve ecological transport of biomass with reduced costs [1, 4]. However, one of the main drivers will be the use of sustainable and environmentally friendly principles, to separate, refine and transform biomass into energy, chemicals and materials. In fact, there is already a large research demand on the use of green chemical technologies in order to minimize the environmental footprints of the end-products [5, 9]. This
chemistry will be a partner for a sustainable future.
1.2 Portuguese agro-forest industries to exploit within the
biorefinery context
Portugal has in agro-forest industries one of the main economic sectors, with pulp and paper and cork industries playing a relevant position. Additionally, the pulp and paper industry is one of the most important industries in world; in fact about 300 million tonnes of paper and paperboard are produced worldwide every year [13]. Eucalyptus spp. are among the most important fiber sources for pulp and paper production [14], with around 19.5 million hectares of Eucalyptus spp. planted worldwide [15]. There are more than 600 species of Eucalyptus, however, only a few are relevant in terms of pulp and paper production. E. globulus is the predominant species planted in Portugal and Spain [16], while E. grandis, which has a tropical origin, is the main species planted in Brazil and South Africa [17]. Other species have been object of interest as raw material for pulp production, such as E. maidenii [18-20] and hybrids, as E. urograndis [21]. Cork, the outer bark of cork oak tree, Quercus suber L., is a unique material. Cork harvesting is a completely natural process, being cork a renewable, sustainable and biodegradable material. Furthermore, the cork oak forest has a great importance on the biodiversity of fauna and flora, being also defended that it has a key role on the protection of several endangered species [22]. Cork industry is one of the main economic sectors in Portugal. It corresponds about 1.7% of the employment and 1.8% of the gross value of production of the manufacturing sector [23]. Furthermore cork products are exported for more than 100 countries, representing about 158 000 tones of exports in 2010, which is equivalent to a value of 755 million Euros [22].
Both Eucalyptus pulping and cork industries generate substantial amounts of biomass residues, among which, bark, in pulp and paper industry, and cork powder and black condensate in cork industry, are the most abundant and are currently simply burned to produce energy. Therefore, the search for new applications is a key strategy in the up-grading of these residues, within the above described biorefinery concept [8]. In fact, there has been an increasing interest in the exploitation of these industrial by-products as a source or precursors of value-added renewable compounds [24-29].
The bark tissue of plants is rich in secondary metabolites, such as phenolic compounds, which have become in recent years one of the most studied groups of
phenolic compounds is related with their antioxidant activity. The increasing interest in the extraction of phenolic compounds from biomass resources, and in particular from industrial by-products [30, 31], is related to two main goals: On one hand, as a way to up-grading industrial by-products and, on the other hand, as a response to the upcoming search to natural products. For instance, in the cosmetic industry, the current new trend is to return to the use of natural plant-derived products.
Obviously, the exploitation of industrial by-products as sources of valuable phenolic compounds has to start by a detailed study of that fraction, with a special concern with the extraction methodologies and techniques of chemical composition analysis used. The conjugation of environmentally friendly extraction procedures with the valorisation of low value biomass residues is of great interest both in the economical and environmental perspectives. Thus, hand in hand with the search of phenolic compounds from agro-forest residues, the use of new sustainable extraction techniques is also a basic requirement. Among several others, supercritical fluid extraction is becoming an increasingly important process to extract high value compounds. Furthermore this technology could be considered as a first step in a biorefinery, allowing to obtain valuable secondary metabolites, without affecting the bulk structure of the biomass feedstock [5, 32].
1.3 Aims and Scope
The detailed study of the chemical composition of pulp and paper and cork industries by-products is a key step towards the implementation of strategies for the recovery of valuable components from these biomass residues. Despite some information have already been reported concerning the phenolic fraction of cork [33-35] and Eucalyptus
globulus bark [36-38], a complete study, mostly applying novel extraction and
characterisation techniques of identification, has not yet been carried out.
In addition, no study has been done so far concerning the industrial cork by-products cork powder and black condensate, as well as, concerning barks from other important
Eucalyptus species, such as E. grandis, E. urograndis and E. maidenii.
Finally, to our knowledge, no study has explored environmentally friendly extraction techniques of phenolic compounds in these biomass resources regarding a future industrial application. In this context several objectives were defined for this thesis:
To explore the advantages of high-performance liquid chromatography and mass spectrometry in the identification and quantification of phenolic compounds;
To select an appropriate method to analyse the antioxidant activity of the extracts obtained;
To characterise the phenolic fractions of E. globulus, E. grandis, E. urograndis and
E. maidenii barks;
To optimise environmentally friendly extraction techniques, namely supercritical carbon dioxide (CO2) in the extraction of phenolic compounds from E. globulus
bark;
To characterise the phenolic fraction of cork;
Finally, to characterise the phenolic fraction of cork powder and black condensate from the cork processing industry.
1.4 Outline of this thesis
This thesis is organized in six chapters. Following the introduction in Chapter 1:
Chapter 2 is a review of the most relevant literature, which includes a description of the cork and pulp and paper industries, evidencing the by-products generated there. A general description of Eucalyptus bark and cork composition is also presented (Part A). The second part of this chapter describes the existing different families of phenolic compounds, the main health benefits attributed to them, as well as a compilation of the different methodologies and techniques used in the analysis of phenolic compounds from vegetal sources (Part B).
Chapter 3 is divided in two parts. In Part A the study of the chemical composition of the phenolic fraction of E. globulus bark is presented. Two different conventional solid-liquid extraction conditions were used, and the respective analysis, involving the use of two different mass spectrometry techniques. Part B of this chapter is focused on the optimisation of supercritical CO2 extraction of phenolic compounds from E. globulus
bark. The optimal SFE conditions are also provided. The content of this chapter was adapted from two papers published in peer-reviewed journals.
Eucalyptus species; E. urograndis, E. grandis and E. maidenii. The optimal solid-liquid
extraction methodology achieved for E. globulus bark was applied here. Once more, the content of this chapter was adapted from a paper published in a peer-reviewed journal.
Chapter 5 describes the characterisation of phenolic fraction of cork as well as of the industrial cork by products, cork powder and black condensate. This chapter was divided in two parts. Part A concerns the analysis of the phenolic fraction of Quercus
suber cork, using a previously described extraction methodology. A second extraction
methodology was also applied. In Part B the extraction methodology used in
Eucalyptus barks was applied to cork and related by-products, namely industrial cork
powder and black condensate. The content of this chapter was adapted from a published paper (Part A) and from a submitted manuscript (Part B), both in peer-reviewed journals.
The adaptation of these chapters from articles involved the standardization of the formatting and nomenclature. Some sentences and images were added, in order to clarify some aspects. In some of the chapters a conclusion part was also added, when it was not part of the original paper.
Chapter 6 presents a general discussion of all the results obtained, representing not only a summary of the main results obtained as also a global comparative analysis of the different results. Final remarks and recommendations for future research activities are also presented in this chapter.
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Chapter 2
Part A
Quercus suber L. cork and
Eucalyptus bark
2.1 Cork and pulp and paper industries
Portugal has in agricultural and forest domains one of the main economic sectors. Besides the dimension of the country, 39% of the area of Portugal is occupied by forest, corresponding about 3 500 000 hectares [1]. Most of this area is occupied by pine, cork oak and eucalyptus plantations (Figure 2.1).
Figure 2.1 – Representative scheme of the forestry occupation by different tree classes in Portugal (source [1])
These trees are the main raw materials of two of the most important industries of the country: the cork and the pulp and paper industries.
2.1.1 Eucalyptus and pulp and paper processing
Pulp, mainly composed of cellulose fibers, is the raw material for paper production and has its origin mostly in plants and particularly in hardwood trees, due to its availability, growing feature and economical and technical factors. Between them, Eucalyptus species are the main fiber sources, due to their fast growing, short rotation periods and favourable pulping and bleaching ability [2]. Eucalyptus plantation areas cover about 19.5 million hectares worldwide [3]. Most of the planted eucalypts are from the subgenus Symphyomyrtus, sections Latoangulatae (E. grandis E. urophylla), Maidenaria (E.
globulus, E. nitens) and Exsertaria (E. camaldulensis, E. tereticornis), including hybrids of
some of these species [4].
Maritime Pine 27% Eucalyptus 23% Cork Oak 23% Holm Oak 13% Oak 5% Stone Pine 4% Sweet Chestnut Tree 1% hardwoodsVarious 3% Various sof twoods 1% Acacias 0%
Eucalyptus globulus Labill. (Figure 2.2) is the main raw material for pulp and paper
industries in Portugal and Spain [5]. There is about 672 000 hectares of E. globulus planted in Portugal, ranking the third in terms of forest area and representing about 31% of the world production of E. globulus [5].
Figure 2.2 – Illustration of Eucalyptus trees, from left to right: E. globulus, E. grandis, E. urograndis and E. maidenii
E. grandis (Figure 2.2) is the most cultivated specie for industrial purposes, particularly in
South Africa and Brazil [2]. Due to its pulping, bleaching and papermaking properties, this specie is one of the main used as fiber source not only in those countries but also in Congo and China [6]. Furthermore, due to its genetic characteristics it is also commonly used in the development of hybrids.
E. urograndis (Figure 2.2), which is a hybrid between E. grandis and E. urophylla, is
produced in Brazil, being developed to conjugate the high density and superior pulp properties of E. urophylla wood and fast growing properties of E. grandis [7]. This demonstrates the increasing interest on the exploitation of Eucalyptus spp. as raw material for pulp production and papermaking in the South America. Actually, during the last 5 years, the eucalyptus forest area in Brazil has increased 5.3% per year, becoming the 6th world pulp producer in 2010 [8].
E. maidenii (Figure 2.2) is, at the moment, not so commonly used as a fiber source for
pulp production as others Eucalyptus species, however, its potential for forest developing and excellent pulp qualities has also been demonstrated [9]. In fact, several countries, as Argentina [10] or even China [11] have shown an increasing interest in to considerer E.
The pulp industries generate substantial amounts of biomass residues, among which bark is the most abundant and is currently simply burned to produce energy. In the case of E.
globulus, bark represents about 11% of the stem dry weight [12]. Thus, a pulp mill with a
production capacity of 5.0x105 tonnes/year of bleached kraft pulp can generate around 1.0x105 tonnes/year of bark, showing the enormous potential for the upgrading of this biomass residue. Apart from the bark residues produced in the mill, large amounts of other biomass residues such as leaves, branches and fruits, resulting from logging operations, are also burned in biomass boilers for energy production or are simply left in the forest for soil nutrition.
2.1.2 Cork, cork oak and cork processing
Cork is the outer bark of Quercus suber L. (Figure 2.3), which is a native tree from Mediterranean region, and occupies in Portugal about 23% of the forestry area, representing more than 700 000 ha [1, 13]. Due to its characteristic climate, dry summers and mild winters, Alentejo is the region with the highest cork oak forestland concentration (about 72% of the total area occupied by this species in Portugal) [13].
Q. suber is harvested at a minimum legal periodicity of 9 years [14]. The unique properties
of this species are related with its capacity to regenerate bark, forming new cork layers. The first cork removing (virgin cork) is only made in trees with 15-30 years old, and each cork oak could reach about 200 years old. The higher quality of cork is only achieved after the third harvesting, from which the cork is called “amadia” grade
Cork presents very interesting and unique properties, such as a low density (with values between 120 and 180 Kg m-3 for amadia grade), hydrophobic character, viscoelastic behaviour and thermal, acoustic and electric insulation properties [15-17].
Portugal is the main exporter of cork based products in the world (53%), resulting from the activity of about 700 industries of this sector, 75% of which located in Aveiro region (Santa Maria da Feira).
The main products of the cork processing are the well-known cork stoppers (Figure 2.3) for wine and champagne bottles, representing about 40% of the production process [13]. However, due to its physical and mechanical properties described above, there are a large number of other cork applications. Several types of agglomerates, insulation cork boarding, flooring and walls coverings or rubber/cork based agglomerates are some of other products from cork processing [14, 16]. In the last decade there was also a high
demand for the use of cork for furniture, footwear, clothe and even some high-tech in aeronautics applications.
Figure 2.3 – a) Cork oak forest, known as “montado” b) cork oak tree c) cork planks d) cork stoppers (source [13])
During cork processing, several residues are generated, called industrial cork powder, black condensate and cooking wastewaters. A representative scheme of the industrial cork processing is illustrated in Figure 2.4. Cork powder, the most abundant cork by-product, represents in Portugal about 34 000 ton per year [18]. This residue is generated during the trituration process to obtain cork granulates (Figure 2.4), which uses the wastes generated during the cork stoppers production, virgin cork and low quality amadia cork [16]. The low particle size of cork powder (less than 0.25 mm) do not allow its use in current industrial uses, even in the agglomerates production, which would entail the use of high amounts of adhesives to cope with its high specific area [16, 18].
Some studies were already developed concerning valorisation of industrial cork powder, such as in the production of composites [16] or in environmental adsorption technologies [19], however, none of these has been industrialized. Consequently, this residue has so far a low commercial value, being mostly used in the biomass boilers to produce energy [18].
Black condensate results from the insulation corkboard production process (Figure 2.4), which uses black agglomerates at high pressures and temperatures in the range of
250-a) b)
c)
500 ºC without any adhesive [18]. During this process vapors are formed, which after cooling generate a waxy solid waste in autoclave pipes. This residue is collected periodically, in amounts of about 2 100 tonnes per year, being also burned to produce energy [18]. Cooking wastewaters, another cork residue, is obtained as a liquid effluent during the cooking of the cork in boiling water.
Figure 2.4 – Representative scheme of the industrial cork processing (adapted from [16])
2.2 Cork and Eucalyptus bark composition
2.2.1 General barks structure
The bark, which protects the trees against physical and biological attacks, constitutes the outer part of woody stem, branches and roots and can be divided in inner and outer bark [20, 21]. The formation of bark is initialised by the cell division of cambium, where the growth of tree occurs. Here xylem is produced, in the woody side and phloem, also known as the inner bark. This is a narrow tissue in which the sap with carbohydrates moves up and down through sieve tubes and rays, transporting the nutrients between the leaves,
Amadia cork Planks Disks Technical cork stoppers Cork stoppers Byproducts By-products/wastes and pieces Virgin cork Granules Agglomerates Agglomerated cork stoppers Cork f loor coverings Insulation cork products Black condensate Cork powder
needles or roots and the stem and branches. The outer bark is a dead tissue, whose cells already existed in the inner bark [20, 22].
The study of the chemical composition of barks is a complex task, due to its variability between tree species as within the morphological parts of the tree. The cell walls of bark, as in wood, are divided in structural and non-structural components (Figure 2.5).
Figure 2.5 – General chemical composition of barks
The structural components comprise polysaccharides (cellulose and hemicelluloses) and lignin. Cellulose is a linear homopolymer with glucose units linked by glycosidic bonds and could represent about 40-45% of the wood of most species, while in the bark this value decreases to around 20% [22]. Lignin confers rigidity and resistance to the impact and compression [20]. Furthermore bark has another structural component, suberin, which is not present in wood. This is composed by an aromatic-aliphatic cross-linked polyester, which plays an important role on thermal and hydric insulation of bark tissues [23].
The non-structural components of bark, which are soluble compounds, are divided into inorganic and organic components, known as ash and extractives, respectively. These components are generally present in higher amounts in bark than in wood [20]. The inorganic constituents (determined as ash) are metallic salts, predominantly of calcium, potassium and magnesium, while the extractives include aliphatic compounds (fatty acids
Bark
Low molecular weight components Organic compounds Extractives Inorganic compounds Ash Macromolecules
Suberin Lignin Polysaccharides
Cellulose
and alcohols and hydrocarbons), terpenic and phenolic compounds, among other families [20]. The role of these components in the tree is related to protection against herbivores or plagues attacks [24]. Therefore, apart from bark, this group of components can usually only be found along wood in the heartwood section [22].
2.2.2 Eucalyptus bark composition
The chemical composition of the bark from E. globulus concerning the structural and non-structural components has been object of a limited number of studies. The E. globulus bark composition (Table 2.1) varies among samples, which could be related with the different origins, age of the tree or morphological part from which the bark was harvested. Only recently the content of suberin of E. globulus bark was reported [25, 26], which is about 1%. Miranda et al. [27] reported that the cellulose content of E. globulus bark (56.0%) is quite similar to wood (56.9%). In fact, the main difference between the two tissues is the extractives content: the bark showed extractives content two times higher than wood [27]. Controversially, earlier Pereira et al. [28] have reported very similar extraction yield values for wood and bark (around 8%).
Table 2.1 – Summary of the global chemical composition (%) of E. globulus, E. grandis and E. urograndis barks
Suberin Lignin Polysaccharides Extractives Ash Source
E. globulus - 22.3 62.8 8.0 1.0 [28] - 16.7 62.5 12.4a 4.74 [29] 1.0 22.3 67.2 7.2 12.8 [25] - 16.9 79.7 6.6 2.9 [27] 1.0 26.6 62.6 6.5 12.1 [26] E. grandis - 11.4 51.0 26.6 7.1 [30] E. urograndis - 16.9 50.8 25.8 4.1 [30] a
sum of the extraction yields with n-hexane, ethanol, methanol and water
It is worth mentioning the fact that the polar extractive components in E. globulus bark are more abundant than the non-polar components. Mirra [25] reported extraction yields of about 4.58% and 1.43% with water and ethanol solid-liquid extractions, respectively, while with dichloromethane only an extraction yield of 0.96% was achieved. Vázquez et al. [29]
also reported a low extraction yield with n-hexane (0.42%), whilst with polar solvents, such as water or a methanol/water mixture, the extraction yield reaches values of 6.80 and 5.19%, respectively. A difference in the lipophilic content of the extractives components between the inner and outer bark of E. globulus was reported [31]. Concerning the inorganic non-structural components, Mirra [25] described a content of about 12.8%, however, most of the studies reported ash content values lower than 5% [27-29].
Information about the contents of structural and non-structural components of E. grandis,
E. urograndis and E. maidenii barks is extremely scarce. In fact, only Bargatto reported, in
his thesis [30], the global chemical composition of E. grandis and E. urograndis. Lower values of polysaccharides and lignin were achieved, comparing with E. globulus composition (Table 2.1). An extremely high value of extractives content was reported for these two species, 26.6% and 25.8% for E. grandis and E. urograndis, respectively [30]. Regarding the chemical composition of the bark from E. grandis, E. urograndis and E.
maidenii, this is also largely an unexplored topic. In fact, only information concerning the
lipophilic fraction of the non-structural extractives components, obtained with dichloromethane, has been found [32]. However, this information is enough to evidence the differences in the chemical composition between these species. Notwithstanding, variations in the lipophilic content between the inner and outer barks of those species have also been observed. E. maidenii, both in the inner and outer bark, has a higher lipophilic content than E. globulus [31], E. grandis and E. urograndis, with values of 2.6 and 6.1%, respectively [32]. E. grandis and its hybrid E. urograndis have lipophilic extraction yields in the same range, with values in the inner bark of 0.3 and 0.5% and in the outer bark of 1.7 and 1.3%, in that order [32]. These two species present a lipophilic content in the outer bark higher than E. globulus, however, in the inner bark the values are similar [31, 32].
The extractives components of the bark of Eucalyptus spp., together with its colour, are the main responsible of the disadvantages in the use of this tissue of the tree in the pulp production [22].
2.2.2.1 Polysaccharides
Polysaccharides comprise mainly cellulose and hemicelluloses in woody materials. Cellulose is a linear macromolecule composed by β(1→4) linked -glucopyranose units (Figure 2.6) [20, 23]. Cellulose contents range between 41.6 [29] and 54.9% [28] in E.
Figure 2.6 – Cellulose structure
Hemicelluloses are heteropolymers composed by different monosaccharides (Figure 2.7), including pentoses (xylose, arabinose) and hexoses (glucose, mannose, galactose, rhamnose, glucuronic acid), sometimes with functionalities, such as methyl or acetyl groups. Furthermore, hemicelluloses have a degree of polymerization lower than cellulose and branched chains [20, 23].
Figure 2.7 – Polysaccharide repetitive monomers
The polysaccharides content of E. globulus bark ranges between 62.5 [29] and 79.7% [27], being this fraction composed mainly by glucose and xylose (Table 2.2). Bargatto [30] reported the polysaccharides composition of E. grandis and E. urograndis with glucose contents of 77.6 and 76.4% and xylose contents of 16.9 and 18.9%, respectively (Table 2.2). Taking the ratio between glucose and xylose as indicative of cellulose to hemicelluloses, there is a significative difference between the three species: a ratio about 3 for E. globulus against 4.58 for E. grandis and 4.04 for E. urograndis. No information has been found about the polysaccharides content of E. maidenii bark.
Table 2.2 – Relative monosaccharides content (%) of E. globulus, E. grandis and E. urograndis barks
Monosaccharides E. globulus E. grandis E. urograndis
[29] [26] [30] [30] Glucose 70.4 68.4 77.6 76.4 Mannose 1.4 1.9 Galactose 3.6 3.3 2.3 1.8 Rhamnose - 0.4 0.7 0.6 Xylose 20.8 23.2 16.9 18.9 Arabinose 3.7 2.7 2.2 2.0 2.2.2.2 Lignin
Lignin is a polyphenolic cross-linked and amorphous polymer built up from phenylpropane units, such as p-coumaroyl, coniferyl and synapil alcohols, which are linked by ether and carbon-carbon bonds (Figure 2.8). The aromatic core of these units is denominated by p-hydroxyphenyl, guaiacyl and syringyl units, respectively [20].
Figure 2.8 – Lignin precursors
The relative abundance of each unit is dependent of each species. To our knowledge, no study concerning the lignin composition of Eucalyptus spp. bark has been published so far.
2.2.2.3 Suberin
Suberin consists of a cross-linked amorphous polyester structure with long chain fatty acids, hydroxy fatty acids and phenolic compounds, linked by esters bonds [33, 34]. The detailed composition of the monomeric composition of suberin, as well as how the monomeric units are assembled at a macromolecular level, is not yet completely understood. Moreover, in the case of Eucalyptus bark, the analysis of suberin remains an unexploitable field. Actually, only recently the suberin content for E. globulus bark was reported [26, 27] and corresponds to a particularly lower value of the bark composition
